Host Cells with Artificial Endosymbionts

ABSTRACT

The present invention is directed generally to eukaryotic cells comprising single-celled organisms that are introduced into the eukaryotic cell through human intervention and which transfer to daughter cells of the eukaryotic cell through at least five cell divisions, and methods of introducing such single-celled organisms into eukaryotic cells. The invention also provides methods of using such eukaryotic cells. The invention further provides single-celled organisms that introduce a phenotype to eukaryotic cells that is maintained in daughter cells. The invention additionally provides eukaryotic cells containing magnetotactic bacteria.

CROSS-REFERENCES TO RELATED APPLICATION(S)

This application is a continuation-in-part of U.S. nonprovisionalapplication Ser. No. 13/74,799, filed on Jan. 13, 2012, and claimspriority to PCT/US2013/021414, filed on Jan. 14, 2013, the entirecontents of which applications are hereby incorporated by reference inits entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates generally to the field of endosymbiosis,artificial endosymbionts, and magnetotactic bacteria. In particular, theinvention provides single-cell organisms such as artificialendosymbionts including magnetotactic bacteria, eukaryotic cells to hostthose single-celled organisms, methods of using eukaryotic cellscontaining single-celled organisms, and methods of introducing thesingle-celled organisms into the eukaryotic cells.

BACKGROUND OF THE INVENTION

Mitochondria, chloroplast and other membrane bound organelles addheritable functionalities, such as photosynthesis, to eukaryotic cells.Such organelles (identified by their vestigial circular DNA) arebelieved to be endosymbiotically derived.

Bacteria exist with a wide range of functionalities not present invarious eukaryotic cells. For example, in 1975 Blakemore identifiedmagnetotactic bacteria (MTB) that orient and swim along a geomagneticfield. (Blakemore, R. Magnetotactic bacteria. Science 24: 377-379 (1975)(which is incorporated by reference in its entirety for all purposes)).These magnetotactic bacteria produce magnetic structures calledmagnetosomes that are composed of magnetite (Fe₃O₄) or greigite (Fe₃S₄)enclosed by a lipid membrane. (Id). A large number of MTB species havebeen identified since their initial discovery. (Id).

Magnetotactic bacteria have been used to selectively bind to andseparate substances. (U.S. Pat. No. 4,677,067 (which is incorporated byreference in its entirety for all purposes)). Additionally, attemptshave been made to add magnetic functionality to cells through externaltags. (Swiston, A. J., Cheng, C., Soong, H. U., Irvine, D. J., Cohen, R.J., Rubner, M. F. Surface Functionalization of Living Cells withMultilayer Patches. Nano Lett. 8(12): 4446-53 (2008) (which isincorporated by reference in its entirety for all purposes)). Bacterialmagnetite has also been introduced into red blood cells by cell fusion(Matsunaga, T., Kamiya, S., (1988), In: Atsumi, K., Kotani, M., Ueno,S., Katila T., Williamsen, S. J. (eds) 6th International Conference onBiomagnetisms (1987). Tokyo Denki University Press, Tokyo, pp. 50-51(which is incorporated by reference in its entirety for all purposes)),and MTB have been introduced into granulocytes and monocytes byphagocytosis. (Matsunaga, T., Hashimoto, K., Nakamura, N., Nakamura, K.,Hashimoto, S. Phagocytosis of bacterial magnetite by leucocytes. AppliedMicrobiology and Biotechnology 31(4): 401-405 (1989) (which isincorporated by reference in its entirety for all purposes)). However,none of these alterations are heritable to daughter cells.

It is an object of the present invention to provide eukaryotic cellscontaining a single-celled organism that is introduced into theeukaryotic cell through human intervention which transfers to daughtercells of the eukaryotic cell through at least five cell divisions, andwhich maintains sufficient copy number in the daughter cells so that adesired functionality introduced by the single-celled organism ismaintained in the daughter cells. It is further an object of the presentinvention to provide eukaryotic host cells containing artificialendosymbionts that are heritable to daughter cells and methods of usesthese eukaryotic cells. It is also an object of the present invention toprovide methods of introducing artificial endosymbionts into the cytosolof eukaryotic host cells. It is another object of the present inventionto provide eukaryotic cells with a heritable magnetic phenotype. It isalso an object of the invention to provide methods of tracking,localizing, or damaging eukaryotic cells.

SUMMARY OF THE INVENTION

The present invention relates to eukaryotic cells comprisingsingle-celled organisms, such as artificial endosymbionts, methods ofusing such eukaryotic cells, and methods of introducing suchsingle-celled organisms into eukaryotic cells. In one embodiment, thesingle-celled organism provides the eukaryotic cell with a desiredfunctionality. In one embodiment, the single-celled organisms areartificial endosymbionts heritable to daughter cells. In anotherembodiment, the artificial endosymbiont is a magnetotactic bacterium. Inone embodiment, the magnetotactic bacterium provides the eukaryotic cellwith a magnetic functionality. In one embodiment, a method of use is amethod of detecting the eukaryotic cells. In another embodiment, amethod of use is a method of magnetically manipulating or targeting theeukaryotic cells. In another embodiment, a method of use is a method ofdamaging the eukaryotic cells.

The artificial endosymbiont of the invention may be modified bydeleting, adding, and/or mutating at least one gene whereby theartificial endosymbiont acquires a trait useful for endosymbiosis orbiotrophy. The genes to be mutated, added, and/or deleted in theartificial endosymbiont may be genes encoding components of theflagellar assembly and genes encoding enzymes for synthesizing essentialmacromolecules, such as amino acids, nucleotides, vitamins, andco-factors. In certain embodiments, the MTB may further be modified toexpress an antibiotic resistance gene or other selectable marker.

In some embodiments the eukaryotic cells of the invention are mammalian,such as mouse, rat, rabbit, hamster, human, porcine, bovine, or canine.In another embodiment the artificial endosymbiont is transmitted fromthe host cell to daughter progeny host cells. In another embodiment, themethod further comprises deleting, inserting, and/or mutating at leastone gene from the eukaryotic cell.

The single-celled organisms of the invention can be introduced intoeukaryotic cells by a number of methods known to those of skill in theart including, but not limited to, microinjection, natural phagocytosis,induced phagocytosis, macropinocytosis, other cellular internalizationprocesses, liposome fusion, erythrocyte ghost fusion, orelectroporation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows positive contrast generated with a T₁ pulse sequence over alog scale concentration up to ˜10⁸ MTB/mL for gfp⁺AMB suspended in agarplugs using a 1.5 T instrument to optimize and characterize the imagingproperties.

FIG. 2 shows a blastula stage mouse embryo that has had one of its twocells at the 2-cell embryo stage microinjected with gfp⁺AMB. The embryois imaged with Leica SP2 AOBS spectral confocal inverted microscopesurrounded by an environmental control chamber for live-cell imagingwith 20×, 0.7 NA objective, and optical zoom of 3×. Panel A showsdifferential interference contrast (DIC) image and Panel B shows a grayscale fluorescence capture of the same image.

FIG. 3 shows the change of total embryo GFP fluorescence of four mouseembryos over time as measured by confocal microscopy. One of the twocells from the 2-cell stage of each embryo had been microinjected withgfp⁺AMB, and the total GFP fluorescence of each embryo was measuredbeginning at the 8-cell stage, 24 hours after microinjection.

DETAILED DESCRIPTION OF THE INVENTION

The invention is illustrated by way of example and not by way oflimitation. It should be noted that references to “an” or “one” or“some” embodiment(s) in this disclosure are not necessarily to the sameembodiment, and all such references mean at least one.

The present invention is directed to eukaryotic cells containingsingle-celled organisms, such as host cells containing artificialendosymbionts in the cytosol of the host cell, and methods ofintroducing the single-celled organism into the eukaryotic cell. In oneembodiment the single-celled organism is an artificial endosymbiont thatis genetically altered. In some embodiments the single-celled organismsare magnetotactic bacteria (MTB).

DEFINITIONS

As used herein, the term “AMB” refers to Magnetospirillum magneticumstrain AMB-1.

As used herein, the term “artificial endosymbiont” refers to asingle-celled organism that is or has been introduced into the cytosolof a eukaryotic cell through human intervention, which has been or canbe transferred to daughter cells of the eukaryotic cell through at leastfive cell divisions, and which maintains sufficient copy number in thedaughter cells so that a phenotype introduced by the artificialendosymbiont is maintained in the daughter cells.

As used herein, the term “cellular life cycle” refers to series ofevents involving the growth, replication, and division of a eukaryoticcell. It is divided into five stages, known as G₀, in which the cell isquiescent, G₁ and G₂, in which the cell increases in size, S, in whichthe cell duplicates its DNA, and M, in which the cell undergoes mitosisand divides.

As used herein, the term “cytosol” refers to the portion of thecytoplasm not within membrane-bound sub-structures of the cell.

As used herein, the term “daughter cell” refers to cells that are formedby the division of a cell.

As used herein, the term “essential molecule” refers to a moleculeneeded by a host cell for growth or survival.

As used herein, the term “genetically modified” refers to altering theDNA of a cell so that a desired property or characteristic of the cellis changed.

As used herein, the term “host cell” refers to a eukaryotic cell inwhich an artificial endosymbiont can reside.

As used herein, the term “liposome mediated” refers to artificialmicroscopic vesicles consisting of an aqueous core enclosed in one ormore lipid layers, used to convey artificial endosymbionts to hostcells.

As used herein, the term “magnetosome” refers to particles of magnetite(i.e., Fe₃O₄) or greigite (Fe₃S₄) enclosed by a sheath or membrane,either as individual particles or in chains of particles.

As used herein, the term “magnetotactic bacteria” or “MTB” refers tobacteria with genes encoding magnetosomes.

As used herein, the term “mammal” refers to warm-blooded vertebrateanimals all of which possess hair and suckle their young.

As used herein, the term “microinjection” refers to the injection ofartificial endosymbionts into host cells.

As used herein, the term “tagged artificial endosymbiont” refers toartificial endosymbionts that have a ligand on the surface of theendosymbiont.

As used herein, the term “parent cell” refers to a cell that divides toform two or more daughter cells.

As used herein, the term “receptor mediated” refers to a molecularstructure or site on the surface of a host cell that binds with anartificial endosymbiont or a tagged artificial endosymbiont followed byinternalization of the artificial endosymbiont.

Artificial Endosymbionts

Single-celled organisms of the invention include bacteria that arecapable of surviving in a eukaryotic cell and maintain copy number suchthat the phenotype introduced by the single-celled organism ismaintained in daughter cells. In some embodiments, the single-celledorganism does not kill the eukaryotic host cell without further humanintervention. In some embodiments, the single-cell organism has afunctionality that is acquired by the eukaryotic cell following theintroduction of the single-celled organism. In some embodiments, thefunctionality of the single-cell organism is magnetism, production of anutrient, desalinization, photosynthesis, or tolerance to harshenvironmental challenges. Magnetism includes diamagnetism andparamagnetism. In some embodiments, the eukaryotic cell maintains thefunctionality for at least 48 hours. In some embodiments, thesingle-celled organism can stably maintain phenotype in the eukaryoticdaughter cells through at least 3 cell divisions, or at least 4division, or at least 5 divisions, or at least 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 cell divisions. In another embodiment,the single-celled organism can stably maintain phenotype in theeukaryotic daughter cells through 3-5 divisions, or 5-10 divisions, or10-15 divisions, or 15-20 divisions.

In an embodiment of the invention, the single-celled organisms of theinvention are genetically modified. Methods for genetically modifyingbacteria are well known in the art. Typically, the bacteria will begenetically modified to improve their survival in eukaryotic host cells,and/or to reduce the toxicity of the single-celled organism to theeukaryotic cell, and/or to provide the eukaryotic cell with a usefulphenotype. In one embodiment, the flagellar proteins of a single-celledorganism are modified so that the single-celled organism no longerexpresses flagellar proteins in the eukaryotic host cell. In anotherembodiment, the single-celled organism is modified so that it can nolonger synthesize an essential molecule that is preferably provided bythe eukaryotic host cell. In an embodiment, the single-celled organismis genetically modified so that its cell cycle is coordinated with thecell cycle of the eukaryotic host cell so that copy number of thesingle-celled organism can be maintained at a sufficient level to impartthe phenotype to daughter cells.

Embodiments of the invention include singled-celled organisms that areProteobacteria. Embodiments of the invention include single-celledorganisms that are α-Proteobacteria. In the current taxonomic schemebased on 16S rRNA, α-proteobacteria are recognized as a Class within thephylum Proteobacteria, and are subdivided into 7 main subgroups ororders (Caulobacterales, Rhizobiales, Rhodobacterales, Rhodospirillales,Rickettsiales, Sphingomonadales and Parvularculales). (Gupta, R. S.Phylogenomics and signature proteins for the alpha Proteobacteria andits main groups. BMC Microbiology, 7:106 (2007) (which is incorporatedby reference in its entirety for all purposes)).

A large number of α-proteobacterial genomes that cover all of the maingroups within α-proteobacteria have been sequenced, providinginformation that can be used to identify unique sets of genes orproteins that are distinctive characteristics of various highertaxonomic groups (e.g. families, orders, etc.) within α-proteobacteria.(Id. (which is incorporated by reference in its entirety for allpurposes)).

Embodiments of the invention include single-celled organisms that aremagnetotactic bacteria (“MTB”). A large number of MTB species are knownto those of ordinary skill in the art since their initial discovery in1975 by Blakemore (Blakemore, R. Magnetotactic bacteria. Science 24:377-379 (1975) (which is incorporated by reference in its entirety forall purposes)) and represent a group of microbes (Faivre, D. & Schüller,D. Magnetotactic bacteria and magnetosomes. Chemistry Reviews 108:4875-4898 (2008) (which is incorporated by reference in its entirety forall purposes)). MTB have been identified in different subgroups of theProteobacteria and the Nitrospira phylum with most of the phylotypesgrouping in α-Proteobacteria. Currently, culturable MTB strains assignedas α-Proteobacteria by 16S rRNA sequence similarity include the strainoriginally isolated by Blakemore in 1975, Magnetospirillummagnetotactium (formerly Aquasprillium magnetotactium), M.gryphiswaldense, M. magneticum strain AMB-1 (“AMB”), M. polymorphum,Magnetosprillum sp. MSM-4 and MSM-6, Magnetococcus marinus, marinevibrio strains MV-1 and MV-2, a marine spirillum strain MMS-1 andMagnetococcus sp. strain MC-1, as well as others. A number of MTB areavailable in pure culture, including AMB. The doubling time of AMB inpure culture is approximately eight hours and is close to that of atypical mammalian cell.

Standard MTB growth media uses succinic acid as the main carbon source,but MTB can be grown with fumarate, tartrate, malate, lactate, pyruvate,oxaloacetate, malonate, P-hydroxybutyrate and maleate as the sole carbonsource. These metabolites are present inside eukaryotic cells.Microaerophillic, facultative anaerobic, and obligate anaerobic MTBstrains have been identified. Oxygen concentrations in the cytosol ofeukaryotic cells are low due to sequestration by proteins such asmyoglobin and concentration in specific cellular locations, e.g.,mitochondria, thus the microaerophilic or facultative anaerobicenvironment necessary for MTB growth is already present in a eukaryoticcell.

MTBs can also be classified by the magnetic particles they synthesize,either magnetite (Fe₃O₄) or greigite (Fe₃S₄). Magnetite producers aremicroaerophilic or facultative anaerobic, need some oxygen source formagnetosome synthesis, and have optimal growth temperatures nearphysiological temperature.

In some embodiments, the single-celled organisms of the invention aregenetically modified. Molecular biology tools have been developed forgenetic manipulations of MTB most extensively in AMB and M.gryphiswaldense strain MSR-1 (reviewed in Jogler, C. and Schtiler, D. inMagnetoreception and Magnetosomes in Bacteria, New York, Springer, 2007p 134-138 (which is incorporated by reference in its entirety for allpurposes)). Since the genome of AMB was the first sequenced of any MTB,all MTB gene references herein refer to this genome unless otherwisespecified. The genomes of two other Magnetospirillum strains andMagnetococcus sp. strain MC-1 have also been recently sequenced. Genesfrom these strains or other MTB strains, presently culturable orunculturable, sequenced or unsequenced, know or unknown, can be used inthe present invention.

The genes responsible for magnetosome formation in MTB cluster ingenomic islands, known as the magnetosome island (MAI). In M.gryphiswaldense, the 130 kb MAI is generally structured into fourpolycistronic operons: the mamAB operon has 17 identified ORFs extendingover 16.4 kb; the mamGFDC operon has 4 identified ORFs is 2.1 kb and 15kb upstream ofmamAB; the mms6 operon has 6 identified ORFs is 3.6 kb and368 by upstream of the mamGFDC; the mamXY operon has 4 identified ORFsis located about 30 kb downstream of mamAB; and the monocistronic mamWgene. In the MA1 the proteins: Mam W, Mgl457, Mgl458, Mgl459, Mms6,Mgl462, MamG, MamF, MamD, MamC, MamH, Maml, MamE, MamJ, MamK, MamL,MamM, MamN, MamO, MamP, MamA, MamQ, MamR, MamB, MamS, MamT, MamU, andMgl505 have been identified, many of which have been given specificfunctions in magnetosome formation. Four genes outside the MAI have beenliked to magnetosome formation, mamY, mtxA, mmsF and mamX. ConservedMAI's have been found in other MTB with some differences in genomicorganization and size.

In some embodiments, genetic modifications are made to the single-celledorganism. Such modifications can be directed modifications, randommutagenesis, or a combination thereof. Natural endosymbionts are donorsof novel metabolic capabilities and derive nutritional requirements fromthe host.

Natural colonization of a host by the symbionts occurs in sevenstages: 1) transmission, 2) entry, 3) countering of host defense, 4)positioning, 5) providing advantage to the host, 6) surviving in hostenvironment, and 7) regulation.

In some embodiments, mutual nutritional dependence (biotrophy) may beestablished between the single-celled organism and the eukaryotic cell.In one embodiment, the single celled organism comprises at least onedeletion of a gene encoding an enzyme for synthesizing an essentialmolecule, wherein said essential molecule is produced by the eukaryotichost cell. An essential molecule can include, but is not limited to, anamino acid, a vitamin, a cofactor, and a nucleotide. For instance,biotrophy can be accomplished by knocking-out the ability of thesingle-celled organism to make an amino acid, which will then be derivedfrom the host. Glycine is a reasonable choice as it is highly abundantin mammalian cells and a terminal product in bacterial amino acidbiogenesis; at least 22 other possibilities exist. The enzyme serinehydroxymethyltransferase converts serine into glycine at the terminus ofthe 3-phosphoglycerate biosynthetic pathway for amino acid production.In one embodiment, the single-celled organism is an AMB in which thegene amb2339 (which encodes the enzyme serine hydroxymethyltransferase)is genetically modified. There are numerous methods for mutating orknocking-out genes known to those of ordinary skill in the art,including in vitro mutagenesis, targeted insertion of DNA into the geneof interest by homologous recombination or deletion of the gene (oroperon, as most of the genes in the bacteria cluster in operons), orusing endonucleases provided appropriate sites only around the targetare present in the genome.

In another embodiment, nutritional dependence for a single-celledorganism on the host cell could also be established by eliminating theability of the single-celled organism to synthesize various metabolites,cofactors, vitamins, nucleotides, or other essential molecules.

In some embodiments of the invention, an MTB has mutations and/ordeletions in genes associated with mobility and/or secretion. MTB areflagellated, and in some embodiments of the invention the MTB has adeletion and/or mutation in at least one gene encoding molecularmachinery associated with the flagella, such that the magnetic bacteriumdoes not produce a functional flagellum. Additionally, many MTB secretevarious compounds, such as hydroxamate and catechol siderophores, whichmay be detrimental to or elicit an immune response from the host. In thesequenced genome of AMB, of the 4559 ORF's, 83 genes have been relatedto cell mobility and secretion. The flagellar assembly is known to becomposed of the gene products of amb0498, amb0500, amb0501, amb0502,amb0503, amb0504, amb0505, amb0610, amb0614, amb0615, amb0616, amb0617,amb0618, amb0619, amb0628, amb1289, amb1389, amb2558, amb2559, amb2578,amb2579, amb2856, amb3493, amb3494 amb3495, amb3496, amb3498, amb3824,and amb3827. The flagella is controlled by the chemotaxis machinerywhich is composed of at least the gene products of amb0322, amb0323,amb0324, amb0325, amb0326, amb1806, amb1963, amb1966, amb2333, amb2635,amb2640, amb2648, amb2652, amb2826, amb2932, amb3002, amb3003, amb3004,amb3007, amb3102, amb3329, amb3501, amb3502, amb3654, amb3879, andamb3880.

In one embodiment, genes encoding antibiotic resistance are insertedinto the genome of the single-celled organism. Eukaryotic cells culturedin media containing the antibiotic will require the single-celledorganism for survival. Neomycin resistance is conferred by either one oftwo aminoglycoside phosphotransferase genes, which also provideresistance against geneticin (G418), a commonly used antibiotic foreukaryotes. Hygromycin B resistance is conferred by a kinase thatinactivates hygromcin B by phosphorylation. Puromycin is a commonly usedantibiotic for mammalian cell culture and resistance is conferred by thepac gene encoding puromycin N-acetyl-transferase. External control ofthe antibiotic concentration allows intracellular regulation of the copynumber of the single-celled organism. Any other system where resistanceor tolerance to an external factor is achieved by chemical modificationof this factor can also be employed. An indirect nutritive advantage oneukaryotic cells may also be established by using MTB and a magneticculture method. In this embodiment, magnetic fields are established toconfer an advantage to eukaryotic cells containing MTB. This could beeither by providing the means for attachment to culture matrix or theaccess to necessary growth or media factors.

In another embodiment, genetic modifications are made the MTB genome toenhance intracellular stability against the host defense mechanisms fora particular host cell type. Many eukaryote endosymbionts andendoparasites, such as the proteobacterial endosymbionts of insects suchas Buchnera, Wigglesworthia, and Wolhachia; the methanogenicendosymbionts of anaerobic ciliates; the nitrogen-fixing symbionts inthe diatom Rhopalodia; the chemosynthetic endosymbiont consortia ofgutless tubeworms (Olavius or Jnanidrillus), the cyanobacterialendosymbionts of sponges, the endosymbionts of all five extant classesof Echinodermata, the Rhizobia endosymbionts of plants, variousendosymbiotic algae, the Legionella-like X bacteria endosymbionts ofAmeoba proteus, numerous Salmonella sp., Mycobacterium tuberculosis,Legionella pneumophila, etc. reside in membrane-bound vacuoles oftentermed symbiosomes, while some species, such as Blochmannia, therickettsia, Shigella, enteroinvasive Escherichia coli, and Listeria,have the ability to inhabit the cytosol. The Dot-Icm Type IV secretorysystem is employed by many intracellular bacteria acquired byphagocytosis to evade the endocytic pathway and persist in the hostcell. This system has been well-studied in L. pneumophila and consistsof the proteins: DotA through DotP, DotU, DotV, IcmF, IcmQ through IcmT,IcmV, IcmW and IcmX. In Photorhabdus luminescens, the luminescentendosymbiont of nematodes, the genes encoding RTX-like toxins,proteases, type III secretion system and iron uptake systems were shownto support intracellular stability and replication. The gene bacA andthe regulatory system BvrRS are essential for maintenance of symbiosisbetween Rhizobia and plants as well as the survival of Brucella ahortusin mammalian cells. The PrfA regulon enables some Listeria species toescape the phagesome and inhabit the cytosol. The desired cellularlocation (e.g., symbiosome or cytosol) of the intracellular MTB willdictate which genes are required to be expressed in the MTB (eitherdirectly from the genome or through a stable vector) for survival andproliferation in the host environment. The endogenous plasmid pMGT ishighly stable in MTB and a number of other broad range vectors (thoseoflncQ, IncP, pBBR1, etc.) are capable of stable replication in MTB.

In another embodiment, the single-celled organism is geneticallymodified by knocking in genes, such as bacteriostatic gene(s),siderophore gene(s), metabolic requirement gene(s), suicide gene(s),life cycle regulation gene(s), transporter gene(s), and escape from thephagosome gene(s). In another embodiment, the single-celled organismsare randomly mutated and subsequently screened for enhanced integrationwithin the host cell. Random mutation can be accomplished by treatmentwith mutagenic compounds, exposure to UV-light or other methods know tothose skilled in the art.

In another embodiment, transgenetic modification(s) are made to countereukaryotic cell defenses using genes from various parasites orendosymbionts. In another embodiment, the population of thesingle-celled organisms in the eukaryotic host cell is regulated thougha balance of intrinsic use of host mechanisms (nutrient availability,control of reproduction, etc.) and antibiotic concentration.

In another embodiment, a natural endosymbiont or an intracellularparasite is genetically modified to produce magnetosomes. Endosymbiontsof insects such as Buchnera, Wigglesworthia, and Wolbachia; themethanogenic endosymbionts of anaerobic ciliates; the nitrogen-fixingsymbionts in the diatom Rhopalodia; the chemosynthetic endosymbiontconsortia of gutless tubeworms (Olavius or Inanidrillus), thecyanobacterial endosymbionts of sponges, the endosymbionts of all fiveextant classes of Echinodermata, the Rhizobia endosymbionts of plants,various endosymbiotic algae, the Legionella-like X bacteriaendosymbionts of Ameoba proteus, numerous Salmonella sp., Mycobacteriumtuberculosis, Legionella pneumophila belong to α-proteobacteria andcould be genetically engineered to produce magnetosomes. In anotherembodiment, a pre-existing organelle can be genetically modified toexpress one or more magnetosome genes to produce an artificialendosymbiont. For instance, mitochondria, plastids, hydrogenosomes,apicoplasts or other organelles, which harbor their own geneticmaterial, can be genetically altered.

In a preferred embodiment, the single-celled organism is an MTB, whichmay or may not be genetically altered, that produces magnetic particlesupon culturing of the eukaryotic cells.

Eukaryotic Cells

The invention provides eukaryotic cells comprising single-celledorganisms in the eukaryotic cells that are heritable and methods ofintroducing the single-celled organisms into host cells.

In some embodiments the eukaryotic cells are plant cells. In someembodiments the eukaryotic cells are cells of monocotyledonous ordicotyledonous plants including, but not limited to, maize, wheat,barely, rye, oat, rice, soybean, peanut, pea, lentil and alfalfa,cotton, rapeseed, canola, pepper, sunflower, potato, tobacco, tomato,eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass, ora forage crop. In other embodiments the eukaryotic cells are algal,including but not limited to algae of the genera Chlorella,Chlamydomonas, Scenedesmus, Isochrysis, Dunaliella, Tetraselmis,Nannochloropsis, or Prototheca, In some embodiments the eukaryotic cellsare fungi cells, including but not limited to fungi of the generaSaccharomyces, Klyuveromyces, Candida, Pichia, Debaromyces, Hansenula,Yarrowia, Zygosaccharomyces, or Schizosaccharomyces.

In some embodiments the eukaryotic cells of the invention are animalcells. In some embodiments the eukaryotic cells are mammalian, such asmouse, rat, rabbit, hamster, human, porcine, bovine, or canine. Miceroutinely function as a model for other mammals, most particularly forhumans. (See, e.g., Hanna, J., Wernig, M., Markoulaki, S., Sun, C.,Meissner, A., Cassady, J. P., Beard, C., Brambrink, T., Wu, L., Townes,T. M., Jaenisch, R. Treatment of sickle cell anemia mouse model with iPScells generated from autologous skin. Science 318: 1920-1923 (2007);Holtzman, D. M., Bales, K. R., Wu, S., Bhat, P., Parsadanian, M., Fagan,A., Chang, L. K., Sun, Y., Paul, S. M. Expression of humanapolipoprotein E reduces amyloid-β deposition in a mouse model ofAlzheimer's disease. J. Clin. Invest. 103(6): R15-R21 (1999); Warren, R.S., Yuan, H., Matli, M. R., Gillett, N. A., Ferrara, N. Regulation byvascular endothelial growth factor of human colon cancer tumorigenesisin a mouse model of experimental liver metastasis. J. Clin. Invest. 95:1789-1797 (1995) (each of these three publications is incorporated byreference in its entirety for all purposes)).

In some embodiments, the eukaryotic cell is a human cancer cell. Thereare many human cancer cell lines that are well known to those ofordinary skill in the art, including common epithelial tumor cell linessuch as Coco-2, MDA-MB231 and MCF7, non-epithelial tumor cell lines,such as HT-1080 and HL60, the NCI60-cell line panel (see, e.g.,Shoemaker, R., The NCI60 human tumor cell line anticancer drug screen.Nature Reviews Cancer 6, 813-823 (2006) (which is incorporated byreference in its entirety for all purposes)). Additionally, those ofordinary skill in the art are familiar with obtaining cancer cells fromprimary human tumors.

In other embodiments, the eukaryotic cells are stem cells. Those ofordinary skill in the art are familiar with a variety of stem celltypes, including Embryonic Stem Cells, Inducible Pluripotent Stem Cells,Hematopoietic Stem Cells, Neural Stem Cells, Epidermal Neural Crest StemCells, Mammary Stem Cells, Intestinal Stem Cells, Mesenchymal stemcells, Olfactory adult stem cells, and Testicular cells.

In an embodiment, the eukaryotic cell is a cell found in the circulatorysystem of a human host. For example, red blood cells, platelets, plasmacells, T-cells, natural killer cells, or the like, and precursor cellsof the same. As a group, these cells are defined to be circulating hostcells of the invention. The present invention may be used with any ofthese circulating cells. In an embodiment, the eukaryotic host cell is aT-cell. In another embodiment, the eukaryotic cell is a B-cell. In anembodiment the eukaryotic cell is a neutrophil. In an embodiment, theeukaryotic cell is a megakaryocyte.

In another embodiment, at least one gene from the eukaryotic cell isgenetically altered. In some embodiments, mutual nutritional dependence(biotrophy) may be established between the artificial endosymbiont andthe eukaryotic cell by genetic modification of the eukaryotic cell,using the appropriate molecular biology techniques specific to thetarget host cell type known to those of ordinary skill in the art,creating eukaryotic cell dependence on the single-celled organism forsome essential macromolecule thus establishing the environmentalpressures for biotrophy. In another embodiment, nutritional dependencefor a single-celled organism on the eukaryotic cell may be establishedby genetically altering the eukaryotic cell to eliminate the ability ofthe single-celled organism to synthesize various metabolites, cofactors,vitamins, nucleotides, or other essential molecules. In suchembodiments, the essential molecule may be provided by the single-celledorganism. In another embodiment, the eukaryotic cell gene encoding theenzyme serine hydroxymethyltransferase, which converts serine intoglycine at the terminus of the 3-phosphoglycerate biosynthetic pathwayfor amino acid production, may be modified.

Methods of Introducing Single-Celled Organisms into Eukaryotic Cells

The single-celled organisms of the invention can be introduced intoeukaryotic cells by a number of methods known to those of skill in theart including, but not limited to, microinjection, natural phagocytosis,induced phagocytosis, macropinocytosis, other cellular uptake processes,liposome fusion, erythrocyte ghost fusion, electroporation, receptormediated methods, and the like. (See Microinjection and OrganelleTransplantation Techniques, Celis et al. Eds.; Academic Press: New York,1986 and references therein, (incorporated by reference in its entiretyfor all purposes)).

In one embodiment, a single-celled organism is introduced to the hostcell by microinjection into the cytoplasm of the host cell. A variety ofmicroinjection techniques are known to those skilled in the art.Microinjection is the most efficient of transfer techniques available(essentially 100%) and has no cell type restrictions (Id.; Xi, Z. &Dobson, S. Characterization of Wolbachia transfection efficiency byusing microinjection of embryonic cytoplasm and embryo homogenate. Appl.Environ. Microbiol. 71(6): 3199-3204 (2005); Goetz, M., Bubert, A.,Wang, G., Chico-Calero, I., Vazquez-Boland, J. A., Beck, M., Slaghuis,J., Szalay, A. A., Goebel, W. Microinjection and growth of bacteria inthe cytosol of mammalian host cells. Proc. Natl. Acad Sci USA98:12221-12226 (2001) (each of these three publications is incorporatedby reference in its entirety for all purposes)).

Naturally phagocytotic cells have been show to take up bacteria,including MTB (Burdette, D. L., Seemann, J., Orth, K. Vibrio VopQinduces PI3-kinase independent autophagy and antagonizes phagocytosis.Molecular microbiology 73: 639 (2009); Wiedemann, A., Linder, S.,Grassi, G., Albert, M., Autenrieth, I., Aepfelbacher, M. Yersiniaenterocolitica invasin triggers phagocytosis via β1 integrins, CDC42Hsand WASp in macrophages. Cellular Microbiology 3: 693 (2001); Hackam, D.J., Rotstein, O. D., Schreiber, A., Zhang, W., Grinstein, S. Rho isrequired for the initiation of calcium signaling and phagocytosis by Fcγreceptors in macrophages. J. of Exp. Med. 186(6): 955-966 (1997);Matsunaga, T., Hashimoto, K., Nakamura, N., Nakamura, K., Hashimoto, S.Phagocytosis of bacterial magnetite by leucocytes. Applied Microbiologyand Biotechnology 31(4): 401-405 (1989) (each of these four publicationsis incorporated by reference in its entirety for all purposes)).

This method is scalable, but may be limited to specific cell types(e.g., macrophage). However, recent studies have shown thatnon-phagocytotic cell types can be induced to endocytose bacteria whenco-cultured with various factors: media and chemical factors, biologicfactors (e.g., baculovirus, protein factors, genetic knock-ins, etc.).(See, e.g., Salminen, M., Airenne, K. J., Rinnankoski, R., Reimari, J.,Valilehto, O., Rinne, J., Suikkanen, S., Kukkonen, S., Yla-Herttuala,S., Kulomaa, M. S., Vihinen-Ranta, M. Improvement in nuclear entry andtransgene expression of baculoviruses by disintegration of microtubulesin human hepatocytes. J. Virol. 79(5): 2720-2728 (2005); Modalsli, K.R., Mikalsen, S., Bukholm, G., Degre, M. Microinjection of HEp-2 cellswith coxsackie B1 virus RNA enhances invasiveness of Shigella flexnerionly after prestimulation with UV-inactivated virus. APMIS 101: 602-606(1993); Hayward, R. D. & Koronakis, V. Direct nucleation and bundling ofactin by the SipC protein of invasive Salmonella. The EMBO Journal 18:4926-4934 (1999); Yoshida, S., Katayama, E., Kuwae, A., Mimuro, H.,Suzuki, T., Sasakawa, C. Shigella deliver an effector protein to triggerhost microtubule destabilization, which promotes Rac1 activity andefficient bacterial internalization. The EMBO Journal 21: 2923-2935(2002); Bigildeev et al. J. Exp Hematol., 39: 187 (2011); Finlay, B. B.& Falkow, S. Common themes in microbial pathogenicity revisited.Microbiol. and Mol. Biol. Rev. 61: 136-169 (1997) (each of these sixpublications is incorporated by reference in its entirety for allpurposes).

The related process, macropinocytosis or “cell drinking,” is a methodnumerous bacteria and viruses employ for intracellular entry (Zhang(2004) In: Molecular Imaging and Contrast Agent Database (MICAD)[database online]; Bethesda (MD): National Library of Medicine (US),NCBI; 2004-2011 (each of these two publications is incorporated byreference in its entirety for all purposes)). Various protocols existwhich can be employed to induce cells to take up bacteria. Severalagents, such as nucleic acids, proteins, drugs and organelles have beenencapsulated in liposomes and delivered to cells (Ben-Haim, N., Broz,P., Marsch, S., Meier, W., Hunziker, P. Cell-specific integration ofartificial organelles based on functionalized polymer vesicles. NanoLett. 8(5): 1368-1373 (2008); Lian, W., Chang, C., Chen, Y., Dao, R.,Luo, Y., Chien, J., Hsieh, S., Lin, C. Intracellular delivery can beachieved by bombarding cells or tissues with accelerated molecules orbacteria without the need for carrier particles. Experimental CellResearch 313(1): 53-64 (2007); Heng, B. C. & Cao, T.Immunoliposome-mediated delivery of neomycin phosphotransferase for thelineage-specific selection of differentiated/committed stem cellprogenies: Potential advantages over transfection with marker genes,fluorescence-activated and magnetic affinity cell-sorting. Med.Hypotheses 65(2): 334-336 (2005); Potrykus (1990) Ciba Found Symp, Vol.1 54: 198 (each of these four publications is incorporated by referencein its entirety for all purposes)). This method is inexpensive,relatively simple and scalable. Additionally, liposome uptake can beenhanced by manipulation of incubation conditions, variation of liposomecharge, receptor mediation, and magnetic enhancement. (See, e.g., Pan etal. Int. J. Pharm. 358: 263 (2008); Sarbolouki, M. N. & Toliat, T.Storage stability of stabilized MLV and REV liposomes containing sodiummethotrexate (acqueous & lyophilized). J. Pharm. Sci. Techno., 52(10):23-27 (1998); Elorza, B., Elorza, M. A., Sainz, M. C., Chantres, J. R.Comparison of particle size and encapsulation parameters of threeliposomal preparations. I Microencapsul. 10(2): 237-248 (1993);Mykhaylyk, O., Sánchez-Antequera, Y., Vlaskou, D., Hammerschmid, E.,Anton, M., Zelphati, O. and Plank, C. Liposomal Magnetofection. MethodsMol. Bio., 605: 487-525 (2010) (each of these four publications isincorporated by reference in its entirety for all purposes)).

Erythrocyte-mediated transfer is similar to liposome fusion and has beenshown to have high efficiency and efficacy across all cell types tested(Microinjection and Organelle Transplantation Techniques; Celis et al.Eds.; Academic Press: New York, 1986 (which is incorporated by referencein its entirety for all purposes)). Typically erythrocytes are loaded byosmotic shock methods or electroporation methods (Schoen, P., Chonn, A.,Cullis, P. R., Wilschut, J., and Schuerrer, P. Gene transfer mediated byfusion protein hemagglutinin reconstituted in cationic lipid vesicles.Gene Therapy 6: 823-832 (1999); Li, L. H., Hensen, M. L., Zhao, Y. L.,Hui, S. W. Electrofusion between heterogeneous-sized mammalian cells ina pellet: potential applications in drug delivery and hybridomaformation. Biophysical Journal 71:479-486 (1996); Carruthers, A., &Melchior, D. L. A rapid method of reconstituting human erythrocyte sugartransport proteins. Biochem. 23: 2712-2718 (1984) (each of these threepublications is incorporated by reference in its entirety for allpurposes). Alternatively, erythrocytes may be loaded indirectly byloading hematopoietic progenitors with single-celled organisms andinducing them to differentiate and expand into erythrocytes containingsingle-celled organisms.

Electroporation is a commonly used, inexpensive method to deliverfactors to cells. (Potrykus, I. Gene transfer methods for plants andcell cultures. Ciba Found Symp 154, 198-208; discussion 208-112 (1990);Wolbank, S. et al. Labeling of human adipose-derived stem cells fornon-invasive in vivo cell tracking. Cell Tissue Bank 8, 163-177 (2007)(each of these two publications is incorporated by reference in itsentirety for all purposes)).

In another embodiment, a eukaryotic cell that naturally endocytosesbacteria (e.g., Chinese hamster ovary (CHO)) is used. In one embodiment,the modified single-celled bacteria are added to the CHO culturedirectly. CHO cells are cultured by standard procedures in Ham's F-12media with 10% fetal calf serum media prior to infection with the MTB.Post infection, the media is augmented with additional iron (40 to 80μM) as either ferric malate or FeCl₃. Numerous other cell typesinternalize bacteria by endocytosis or more specifically phagocytosis;endosymbionts or parasites have their own methods for cellular entry andthese natural processes can be exploited for internalization of theartificial endosymbionts resulting in the generation of so-calledsymbiosomes. In another embodiment, symbiosomes from one cell can betransplanted to another cell type (i.e., one incapable of endocytosis ofartificial endosymbionts) using microinjection, organelletransplantation, and chimera techniques. These host cells are culturedin typical media and with the techniques for the specific cell type.

In one embodiment, a single-celled organism is introduced to the hostcell by a liposome mediated process. Mitochondria and chloroplasts,which are larger than MTB, have been efficiently introduced intoeukaryotic cells when encapsulated into liposomes. (Bonnett, H. T.Planta 131, 229 (1976); Giles, K.; Vaughan, V.; Ranch, J.; Emery, J.Liposome-mediated uptake of chloroplasts by plant protoplasts. In VitroCellular & Developmental Biology—Plant 16(7) 581-584 (each of these twopublications is incorporated by reference in its entirety for allpurposes)). Numerous liposome fusion protocols and agents are availableand can be used by the skilled artisan without undue experimentation.(See, e.g., Ben-Haim, N., Broz, P., Marsch, S., Meier, W., Hunziker, P.Cell-specific integration of artificial organelles based onfunctionalized polymer vesicles. Nano Lett. 8(5): 1368-1373 (2008);Lian, W., Chang, C., Chen, Y., Dao, R., Luo, Y., Chien, J., Hsieh, S.,Lin, C. Intracellular delivery can be achieved by bombarding cells ortissues with accelerated molecules or bacteria without the need forcarrier particles. Experimental Cell Research 313(1): 53-64 (2007);Heng, B. C. & Cao, T. Immunoliposome-mediated delivery of neomycinphosphotransferase for the lineage-specific selection ofdifferentiated/committed stem cell progenies: Potential advantages overtransfection with marker genes, fluorescence-activated and magneticaffinity cell-sorting. Med. Hypotheses 65(2): 334-336 (2005); Potrykus(1990) Ciba Found Symp, Vol. 1 54: 198 (each of these four publicationsis incorporated by reference in its entirety for all purposes)).

Methods of Use of Eukaryotic Cells Comprising Single-Celled Organisms

This invention provides methods of using phenotypes introduced intoeukaryotic cells by single-celled organisms of the invention. In someembodiments, the phenotype used is a heritable functionality nototherwise present in the eukaryotic cells. In some embodiments,eukaryotic cells with a magnetic phenotype are magnetically manipulated.

In some embodiments eukaryotic cells of the invention with magneticphenotypes can be detected and monitored using magnetic detection orimaging techniques such as magnetic resonance imaging (MRI). MRI is awidely used clinical diagnostic tool because it is non-invasive, allowsviews into optically opaque subjects (including mice, humans, and othermammals), and provides contrast among soft tissues at reasonably highspatial resolution, compared to non-magnetic imaging (such as opticalprobes) which tend to have low special resolution and to be limited inpenetration depth. Conventional MRI focuses almost exclusively onvisualizing anatomy and has no specificity for any particular cell type.The ‘probe’ used by conventional MRI is the ubiquitous proton ¹H inmobile water molecules. Contrast agents can be used for cell-typespecificity, but contrast agents dilute or have toxicology issues, andcan only be used for short-term studies. Some embodiments of thisinvention facilitate cell-specific MRI imaging in living subjects forlonger-term studies.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are mammalian cancer cells such as human cancer cell line NCI60, other human cancer cell lines, murine cancer cells lines, or caninecancer cell lines. These magnetic cancer cells can be injected intoimmunocompromised mammals such as mice and can then be monitored withmagnetic imaging to track tumor progression over time. In someembodiments, anti-cancer treatments or putative treatments may beprovided to the immunocompromised mammal during the period that tumorprogression is being tracked in real time. In some embodiments,viability of eukaryotic cells of the invention with magnetic phenotypesis monitored using MRI to assess in vivo cell response to differentconditions, including drug treatments.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are metastatic cancer cells that are introduced intoexperimental animals by methods including injection. MRI can then beused to monitor the process of metastasis and movement of metastaticcancer cells throughout the experimental animals. In some embodimentsmagnetic eukaryotic cancer cells of the invention are injected into atumor bearing mammal, such as a mouse, and MRI is used to trackmetastatic cell circulation through the mammal.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are macrophages and are injected into experimental animals.Magnetic imaging is used to detect any aggregations of macrophageswithin the animals. Macrophages aggregate to the sites of inflammation,which can be caused by malignant lesions including metastasis.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are stem cells or were derived from stem cells, including EScells, iPS cells, or adult stem cells obtained from mammalian speciesincluding but not limited to human, mouse, rat, and pig. Stem cells maybe introduced into a target organism directly or may be firstdifferentiated in vitro and then introduced into a target organism. Thein vivo fate, including localization, growth rates and viability, of theintroduced cells can be assayed through magnetic imaging.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are hematopoietic stem or progenitor cells, which are thenintroduced into a mammal. When hematopoietic stem or progenitor cellsare introduced into mammals, these cells will reside in the bone marrow.The behavior of the magnetic hematopoietic stem or progenitor cells,including their localization, proliferation and mobilization into bloodstream upon receiving different stimuli, can be monitored throughmagnetic imaging.

In some embodiments, magnetic artificial endosymbionts divide moreslowly than stem cell host cells in which they reside. Over time, stemcells, which generally divide more slowly than more differentiatedprogenitor cells, will retain magnetic phenotype longer than moredifferentiated progenitor cells and the two types of cells will becomemeasurably district when imaged magnetically. In some embodiments,eukaryotic cells of the invention with magnetic phenotypes are fused toa eukaryotic cell line of a desired cell type, creating chimeric calls.Chimeric cells can be introduced into an animal and tracked by magneticimaging.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are embryonic cells. In some embodiments the embryonic cellsare fertilized animal eggs, such as mouse or rat. In some embodiments,embryos are implanted into female animals and allowed to develop,leading to the production of animals with cells containing single-celledorganism throughout their bodies. Magnetic tissues can be harvested fromthe resulting organisms and magnetic cell lines can be derived fromthem. In some embodiments, these animals are bred and the magneticphenotype is inherited maternally. In some embodiments, eukaryotic cellsof the invention with magnetic phenotypes are introduced intomulti-celled embryos. The cell lineage of the magnetic cell can betracked by magnetic imagine as the embryo develops.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are moved by magnetically attracting the eukaryotic cells. Insome embodiments, this movement is achieved using externally generatedmagnetic fields and field gradients. Various devices have been reportedfor magnetic targeting, such as those in U.S. Pat. No. 8,159,224 andRiegler J, Liew A, Hynes S O, Ortega D, O'Brien T, Day R M, Richards T,Sharif F, Pankhurst Q A, Lythgoe M F. Superparamagnetic iron oxidenanoparticle targeting of MSCs in vascular injury. Biomaterials. 2013March; 34(8):87-94 In some embodiments, eukaryotic cells of theinvention with magnetic phenotypes are separated from a heterogeneouspopulation of non-magnetic cells, either in vitro or in vivo (followingintroduction into an organism) by using a magnet to attract the magneticcells.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are introduced into the bloodstream or other fluids of atarget organism. The eukaryotic cells can be directed to an area ofinterest on the organism and localized there with an aid of a magnetpositioned adjacent to this area. In some embodiments, the eukaryoticcells can be stem cells that are directed to and held in an area on amammal's body where they could have therapeutic effect. In someembodiments, the eukaryotic cells can be immune cells which can bedirected to a particular location on a mammal's body, including to atumor or injury site. In some embodiments, the eukaryotic cells can beloaded with a therapeutic agent that can be released after beingmagnetically directed to a desired area.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are placed in an alternating magnetic field, or alternativemagnetic field, referred to as an AMF, for a technique called MagneticHyperthermia Technique (MHT). AMF and MHT are described in U.S.Publication No. US20120302819 (U.S. application Ser. No. 13/510,416),and in Silva A C, Oliveira T R, Mamani J B, Malheiros S M, Malavolta L,Pavon L F, Sibov T T, Amaro E Jr, Tannús A, Vidoto E L, Martins M J,Santos R S, Gamarra L F. Application of hyperthermia induced bysuperparamagnetic iron oxide nanoparticles in glioma treatment. Int J.Nanomedicine. 2011; 6:591-603 (both of which are incorporated byreference in its entirety for all purposes). Hyperthermia is atherapeutic procedure that promotes the increase of temperature in bodytissues in order to change the functionality of the cellular structures.Its activity is based on the fact that a temperature increase can inducecell damaged, including cell lysing and cell death. In some embodiments,the eukaryotic cells of the invention are subjected to an AMF for 10,20, 30, 40, 50, 60, 70, 80, or 90 minutes. In some embodiments, magneticfield frequencies of an applied AMF lie between 50 kHz and 1 MHz. Insome embodiments, the magnetic field amplitude of an applied AMF remainsbelow 100 mTIn. In some embodiments, the eukaryotic cells of theinvention subjected to MHT are tumor cells, which are less resistant tosudden increases in temperature than the normal surrounding cells. Insome embodiments, the eukaryotic cells of the invention are tumor cellsor are next to tumor cells and are subjected to an alternating magneticfield until the internal temperature of the tumor reached between 43degrees Celsius and 47 degrees Celsius.

In some embodiments, eukaryotic cells of the invention with magneticphenotypes are placed in a spinning magnetic field, resulting inrotation of the single-celled organisms inside the cells and celldamage, including cell lysing and death. In some embodiments, eukaryoticcells of the invention with magnetic phenotypes in a heterogeneouspopulation of non-magnetic cells are selectively damaged by subjectingthe entire cell population to an alternating magnetic field or to aspinning magnetic field. In some embodiments, eukaryotic cells of theinvention with magnetic phenotypes in a heterogeneous population ofnon-magnetic cells are placed in an alternating magnetic field or aspinning magnetic field, resulting in damage to both the eukaryoticcells of the invention and the non-magnetic cells located near the cellsof the invention. In some embodiments, eukaryotic cells of the inventionwith magnetic phenotypes are introduced into an animal and are targetedto a location within the animal by magnetic manipulation or other formsof cell targeting known in the art. The location within the animal canthen be subjected to an alternating magnetic field or a spinningmagnetic field, resulting in the damage to cells surrounding themagnetic cells. In some embodiments, eukaryotic cells of the inventionwith magnetic phenotypes are introduced into an animal and are localizedto a tumor site within the animal by magnetic manipulation or otherforms of cell targeting known in the art. The tumor can then besubjected to an alternating magnetic field or a spinning magnetic field,resulting in the damage to tumor cells surrounding the magnetic cells.In some embodiments, eukaryotic cells of the invention with magneticphenotypes are stem cells, including ES cells, iPS cells, or adult stemcells obtained from mammalian species including but not limited tohuman, mouse, rat, and pig. Stem cells may be introduced into a targetorganism directly or may be first differentiated in vitro and thenintroduced into a target organism. Following introduction, the animalcan be subjected to an alternating magnetic field or a spinning magneticfield, resulting in the death of introduced stem cells and resultinglineages of these stem cells. The in vivo fate, including localization,growth rates and viability, of the introduced cells can be assayedthrough magnetic imaging.

The inventions disclosed herein will be better understood from theexperimental details which follow. However, one skilled in the art willreadily appreciate that the specific methods and results discussed aremerely illustrative of the inventions as described more fully in theclaims which follow thereafter.

EXAMPLES Example 1 Microinjection of gfp⁺AMB into Murine Cells

A. Construction of gfp⁺AMB.

Expression vectors for eGFP, one including a Shine-Dalgarno sequenceupstream of the gfp gene and one without a Shine Dalgarno, sequence werecloned into cryptic broad host range vector pBBR1MCS-2 (Kovach, M. E.,et al. Four new derivatives of the broad-host-range cloning vectorpBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166,175-176, (1995) (which is incorporated by reference in its entirety forall purposes)). AMB (ATCC 700264) was transformed with this construct.(Matsunaga, T. et al. Complete genome sequence of the facultativeanaerobic magnetotactic bacterium Magnetospirillum sp. strain AMB-1. DNARes. 12, 157-166 (2005); Burgess J. G., et al. Evolutionaryrelationships among Magnetospirillum strains inferred from phylogeneticanalysis of 16S rDNA sequences. J. Bacteriol. 175: 6689-6694 (1993);Matsunaga T, et al. Gene transfer in magnetic bacteria: transposonmutagenesis and cloning of genomic DNA fragments required formagnetosome synthesis. J. Bacteriol. 174: 2748-2753 (1992); Kawaguchi R,et al. Phylogeny and 16s rRNA sequence of Magnetospirillum sp. AMB-1, anaerobic magnetic bacterium. Nucleic Acids Res. 20: 1140, (1992) (each ofthese four publications is incorporated by reference in its entirety forall purposes)).

Transformation was achieved by conjugation using a donor Escherichiacoli strain as described by Goulian, M. van der Woude, M. A. A simplesystem for converting lacZ to gfp reporter fusions in diverse bacteria.Gene 372, 219-226 (2006); Scheffel, A. Schiller, D. The AcidicRepetitive Domain of the Magnetospirillum gryphiswaldense MamJ ProteinDisplays Hypervariability but Is Not Required for Magnetosome ChainAssembly. J. Bacteriol. September; 189(17): 6437-6446 (2007) (each ofthese two publications is incorporated by reference in its entirety forall purposes). The mating reactions were cultured for 10 days underdefined microaerophilic conditions in the absence of DAP to select forpositive transformants.

Following conjugation, gfp⁺AMB transformants with and without theShine-Dalgarno sequence successfully displayed GFP fluorescence. Thetranformants containing the Shine-Dalgarno sequence displayed higherlevels of GFP fluorescence than the transformants without this sequence.The resulting fluorescence did not leave the gfp⁺AMB cells when viewedat 100× magnification at 488 nm excitation.

The magnetic properties of the gfp⁺AMB were analyzed by MRI. The gfp⁺AMBwas suspended in agar plugs using a 1.5 T instrument to optimize andcharacterize the imaging properties. FIG. 1 shows the positive contrastgenerated with a T₁ pulse sequence over a log scale concentration up to˜10⁸ MTB/mL. Signal intensity was related to concentration.

B. Microinjection into Murine Embryonic Cells.

The gfp⁺AMB was mircoinjected into one cell of each of 170 mouse embryosat the 2-cell stage. Six concentrations over a log scale up to ˜10⁵gfp⁺AMB were injected per cell, estimated by the optical density at 565nm. Death rate of cells following microinjection was constant across thedifferent injected concentrations. Images overlaying fluorescent anddifferential interference contrast (DIC) images of cells injected withthe highest concentration (10⁵ MTB/cell) were compared. An uninjectedcontrol exhibited low levels of autofluorescence. Slices at differenthorizontal planes in 8-cell embryos at a given time point were compared.In each embryo, all four cells derived from the injected cell showedsignificant fluorescence while none of the four cells derived from theuninjected internal controls displayed significant fluorescence.

The embryos were allowed to develop for three days after the injection.In each concentration level, embryos survived for up to the full threedays developing to the 256 cell blastula stage and appeared healthyenough for implantation. Numerous cells within each blastula displayedsignificant fluorescence, demonstrating that the artificialendosymbionts were transferred to daughter cells across multiple celldivisions as the embryos comprising the eukaryotic host cells developedto the blastula stage. One such blastula is shown in FIG. 2, where PanelA shows a differential interference contrast (DIC) image of the blastulaand Panel B) shows a gray scale fluorescence capture of the same image,showing fluorescence in numerous cells throughout the blastula.

Confocal microscopy was used to quantify total expression of GFPthroughout four individual embryos by measuring total GFP fluorescencein the entire embryo over time at various points beginning at the eightcell stage of the embryo. FIG. 3 shows the change of embryo fluorescenceover time. This indicates that the copy number of artificialendosymbionts was maintained in daughter cells for at least sevengenerations, such that the fluorescent phenotype of the host cells wasmaintained as the embryo progressed from the 2-cell stage to the256-cell blastula stage.

These results demonstrate that, when delivered by microinjection,gfp⁺AMB were not immediately cleared or degraded and were not toxic tothe developing embryo over the course of the three day experiment.Microinjected embryos divided normally, suggesting that gfp⁺AMB do notdisplay pathogenic markers or secret toxic compounds. They weretransferred to daughter cells across many cell divisions, were containedin the cytoplasm, were punctate and well distributed, and maintainedcopy number within the daughter host cells, such that the fluorescentphenotype of the eukaryote host cells was maintained in daughter cellsthrough at least seven generations. These results demonstrate that AMBcan be stably maintained intracellularly and are transferred to daughtercells over at least seven cell divisions.

Example 2 Phagocytic Entry of AMB

Receptor mediated: The inlAB gene is amplified from L. monocytogenesgenomic DNA (ATCC 19114) and is inserted into pBBR1MCS-5,107 thegentamicin cognate of pBBR1MCS-2 (Kovach, M. E., et al. Four newderivatives of the broad-host-range cloning vector pBBR1MCS, carryingdifferent antibiotic-resistance cassettes. Gene 166, 175-176, (1995)),and gfp+inlAB+AMB is generated. The gfp+inlAB+AMB is co-cultured witheukaryotic host cells, including common epithelial tumor cell linesCoco-2, MDA-MB231 and MCF7, non-epithelial tumor cell lines, such asHT-1080 and HL60, and murine stem cells. Fluorescent microscopy and FACSare used to monitor and quantify internalization and intracellularlocation.

Expression of pore-forming haemolysin (hlyA) in AMB is achieved throughamplification of hlyA from L. monocytogenes genomic DNA (ATCC 19114).The amplified hlyA is inserted into pBBR1MCS-3 (the tetracycline cognateof pBBR1MCS-2) which is then used to transform gfp⁺AMB. The resultingAMB strain is exposed to murine macrophage cell line J774, capable ofspontaneous phagocytosis. Gentomycin treatment is used to eliminatebacteria not internalized and hlyA⁻ AMB is used as negative control.Fluorescent microscopy is used to monitor the intracellular fate andlocalization of AMB.

If bacteria remain confined to the phagosomes, two genes, plcA and plcB,implicated in escape of L. monocytogenes into the cytosol, areintroduced. (Smith, G. A., Marquis, H., Jones, S., Johnston, N. C.,Portnoy, D. A., Goldfine, H. Infection and immunity 63: 4231 (1995);Camilli, A.; Goldfine, H.; Portnoy, D. A. The Journal of ExperimentalMedicine 173: 751 (1991) (each of these two publications is incorporatedby reference in its entirety for all purposes)). If bacteria escapesuccessfully, but fail to propagate, hpt is introduced. (Goetz, M.,Bubert, A., Wang, G., Chico-Calero, I., Vazquez-Boland, J. A., Beck, M.;Slaghuis, J., Szalay, A. A., Goebel, W. Proc Natl Acad Sci USA 98: 12221(2001); Chico-Calero, I., Suarez, M., Gonzalez-Zorn, B., Scortti, M.,Slaghuis, J., Goebel, W., Vazquez-Boland, J. A. Proc Natl Acad Sci USA99: 431 (2002) (each of these two publications is incorporated byreference in its entirety for all purposes)). In L. monocytogenes, hptencodes the transporter responsible for uptake of glucose-6-phosphatefrom the cytosol. Other genes from L. monocytogenes have been implicatedin sustaining growth within host (glnA and gltAB and argD) and these aresystematically introduced as needed. (Joseph, B., Przybilla, K.,Stuhler, C., Schauer, K., Slaghuis, J., Fuchs, T. M., Goebel, W. Journalof Bacteriol. 188: 556 (2006) (which is incorporated by reference in itsentirety for all purposes)).

Example 3 Regulation of AMB Growth

Regulation of AMB growth in embryonic stem cells can be regulated asfollows. Coleoptericin-A (ColA) is amplified from total Sitophilusoryzae cDNA. Expression of ColA in beetles of genus Sitophilus regulatestiters of γ-Protobacterium, which has naturally developed closesymbiotic relationship the beetles, and resides in specific cells calledbacteriocytes. (Login, F. H., Balmain, S., Vallier, A., Vincent-Monegat,C., Vigneron, A., Weiss-Gayet, M., Rochat, D., Heddi, A. Antimicrobialpeptides keep insect endosymbionts under control. Science 334(6054):362-365 (2011) (which is incorporated by reference in its entirety forall purposes)).

Murine embryonic stem cells comprising gfp+AMB are treated using aneural differentiation protocol. MTB expression levels are quantifiedusing qPCR and fluorescent microscopy. Amplified colA is then expressedin the gfp+AMB embryonic stem cells. A promoter is selected to provideoptimal ColA expression levels.

Example 4 Magnetic Pheonotype of Murine Cells Containing gfp⁺AMB

Cells from macrophage cell line J774.2 derived from murine ascites andsolid tumor with introduced gfp⁺AMB were applied to a magnetic columnand were retained by the column. These results demonstrate that,following introduction of gfp⁺AMB, J774.2 murine cells were magneticallydetected and magnetically manipulated, as they were magneticallyconcentrated and magnetically collected.

Example 5 Gfp⁺AMB in Human Breast Cancer Cell Line MDA-MB-231

Gfp⁺AMB, Gfp+InlA/B+AMB, and Gfp+Pla1+AMB were each introduced to humanbreast cancer cells from cell line MDA-MB-231. GFP fluorescence wasdetected in more than 90% of the MDA-MB-231 cells 48 hours after theintroduction of each of Gfp⁺AMB, Gfp+InlA/B+AMB, and Gfp+Plal+AMB. GFPfluorescence in Gfp⁺AMB was observed in these MDA-MB-231 cells at least13 days after introduction of gfp⁺AMB, in the fourth passage of theMDA-MB-231 cells following the introduction, where MDA-MB-231 cellpopulation doubled three to four times between each passage. GFPfluorescence was observed in both forming daughter cells of anMDA-MB-231 cell with introduced gfp⁺AMB in the process of cell division.

Following the introduction of gfp⁺AMB, MDA-MB-231 cells were stainedwith Lysotracker® Red DND-99 dye (specific to lysosomes) and Hoechst33342 nuclear stain purchased from Life Technologies. Green GFPfluorescence was observed as localized within individual MDA-MB-231cells and distinct from red lysosome staining and blue nuclear stainingsuggesting that AMB were localized in cytoplasm and not digested throughlysosome pathway.

At 24 hours and 72 hours after introduction, plated MDA-MB-231 cells andplated control MDA-MB-231 cells were fixed in formalin andglutaraldehyde following a wash with PBS. Cells were then stained withPrussian Blue, and observed by microscopy at 40× magnification. Ironstaining was observed in some of the MDA-MB-231 cells with introducedgfp⁺AMB but not in the control MDA-MB-231 cells without introducedgfp⁺AMB. The proportion of cells displaying iron staining was similarbetween the MDA-MB-231 cells 72 hours after gfp⁺AMB introduction and theMDA-MB-231 cells 24 hours after gfp⁺AMB introduction.

hours after the introduction of gfp⁺AMB, MDA-MB231 cells weretrypsinized and resuspended in PBS. One sample of these cells was placedinto a glass slide chamber. A magnet was aligned to the side of chamberand cell movement was observed under microscope at 20× magnification.MDA-MB231 cells with introduced gfp⁺AMB, but not control MDA-MB231 cellswhich had not had gfp⁺AMB introduced, exhibited movement toward themagnet. Another sample of these cells was placed into small tubes, whichwere taped to a magnet for one hour. Control MDA-MB231 cells which hadnot had gfp⁺AMB introduced settled down at the bottom of the tube.However, MDA-MB231 cells with introduced gfp⁺AMB were aligned to themagnet side of the tubes.

These results indicate that gfp⁺AMB were not immediately cleared fromhuman breast cancer MDA-MB-231 cells. They were transferred to daughtercells across at least 12 cell divisions and were located within theMDA-MB-231 cells outside of both the lysosomes and nuclei. These resultsalso demonstrate that at least 48 hours following introduction ofgfp⁺AMB, the MDA-MB-231 cells containing gfp⁺AMB displayed weremagnetically detected and magnetically manipulated, as they weremagnetically moved, magnetically targeted to a location, magneticallyconcentrated, and magnetically collected. Additionally, at least 72hours following introduction of gfp⁺AMB, the MDA-MB-231 cells containedobservable quantities of iron.

Example 6 Gfp⁺AMB in Human Induced Pluripotent Stem Cells

GFP fluorescence was observed in Human Induced Pluripotent Stem (“IPS”)cells at least eight days following introduction of gfp⁺AMB to the IPScells, in the second passage of the IPS cells. These results indicatethat gfp⁺AMB were not immediately cleared from human IPS cells, and weretransferred to daughter cells.

Example 7 Cell Imaging within Mouse Tumor

Cell visualization was tested in a mouse bearing two subcutaneoustumors, one on its left flank and one on its right flank. 1.5×10⁶MDA-MB231 cells containing introduced gfp⁺AMB were injected directlyinto the tumor on the left flank of the mouse. An equivalent number ofcontrol MDA-MB231 cells without introduced cells were injected on theright flank of the mouse. The mouse was imaged using a bench top IT MRIwith T2w pulse sequences. The resulting image showed a dark area at thetumor on the left side of the mouse, the site of the injection ofMDA-MB231 cells containing introduced gfp⁺AMB, but no signal at thetumor on the right side of the mouse, where control MDA-MB231 cellsinjected into a left side tumor.

Example 8 Monitoring of Mouse Tumor

Gfp⁺AMB cells are introduced into MDA-MB231 human cancer cells. Theresulting magnetic cells and their daughter cells are injected intomammary fat pads of a group of immunocompromized mice. Tumor growth ismonitored at regular intervals by MRI imaging. Mice with establishedtumors assigned either to an experimental group or to a control group.Mice in the experimental group are treated with a potential anti-tumortherapeutic compound while mice in the control group are treated with aninactive vehicle. MRI is used to monitor the size and growth of thetumor non-invasively following the treatments to assess the efficacy ofthe tested compound in combatting the tumor.

Example 9 Magnetic Enhancement of Cell Retention

Gfp⁺AMB cells are introduced into Rat Cardiac-Derived Stem Cells (CDC).The resulting magnetic CDC cells are used in the Ischemia/Reperfusionmodel. Rats are treated as described Cheng K, Malliaras K, Li T S, SunB, Houde C, Galang G, Smith J, Matsushita N, Marban E. Magneticenhancement of cell retention, engraftment, and functional benefit afterintracoronary delivery of cardiac-derived stem cells in a rat model ofischemia/reperfusion. Cell Transplant. 2012; 21(6):1121-35. The magneticCDC cells are then introduced into the left ventricle cavity of thetreated rates. A 1.3 T magnet is placed above the heart during and afterthe injection. The animal's chest is closed and it is allowed torecover. The short and long-term behavior of the labeled CDC in the ratis monitored by MRI imaging at regular intervals.

Example 10 AMF tumor treatment

Gfp⁺AMB cells are introduced into MDA-MB231 cells. The resulting cellsare injected into subcutaneous tumors, formed by 4T1 cells in nude mice.Untreated control MDA-MB231 cells are injected into a tumor at theopposite flank of each animal. Each animal is placed into alternatingmagnetic field (30.6 kA/m, 118 kHz) for 30 minutes, and allowed torecover following the procedure. Animals are sacrificed at regularintervals and histological analyzes are performed on tumors from bothmice with injected magnetic MDA-MB231 and control mice with injectedcontrol MDA-MB231. In the experimental mice, the labeled cells and thesurrounding tumor are damaged leading to damage to the tumor overtime.

All publications, patents and patent applications discussed and citedherein are incorporated herein by reference in their entireties. It isunderstood that the disclosed invention is not limited to the particularmethodology, protocols and materials described as these can vary. It isalso understood that the terminology used herein is for the purposes ofdescribing particular embodiments only and is not intended to limit thescope of the present invention which will be limited only by theappended claims.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method of detecting a eukaryotic cell, comprisingmagnetically imaging a eukaryotic cell containing at least onemagnetotactic bacterium.
 2. The method according to claim 1, wherein theeukaryotic cell is contained within an animal.
 3. The method accordingto claim 2, wherein the animal is a mammal.
 4. The method according toclaim 3, wherein the mammal is murine.
 5. The method according to claim3, wherein the mammal is human.
 6. The method according to claim 3,wherein the eukaryotic cell is a cancer cell.
 7. The method according toclaim 6, wherein the mammal has been treated with a potential anti-tumoragent.
 8. The method according to claim 3, wherein the eukaryotic cellis or was derived from an IPS cell.
 9. A method of magneticallymanipulating a eukaryotic cell, comprising magnetically localizing aeukaryotic cell containing at least one magnetotactic.
 10. The methodaccording to claim 9, wherein the eukaryotic cell is contained in ananimal.
 11. The method according to claim 10, wherein the animal is amammal.
 12. The method according to claim 11, wherein the animal ismurine.
 13. The method according to claim 11, wherein the animal ishuman.
 14. The method according to claim 11, wherein the eukaryotic cellis localized to a tumor.
 15. A method of damaging a eukaryotic cell,comprising subjecting a eukaryotic cell containing at least onemagnetotactic bacterium to an alternating magnetic field or a spinningmagnetic field.
 16. The method according to claim 15, wherein theeukaryotic cell is contained in an animal.
 17. The method according toclaim 16, wherein the animal is a mammal.
 18. The method according toclaim 17, wherein the mammal is murine.
 19. The method according toclaim 17, wherein the mammal is human.
 20. The method according to claim17, wherein the eukaryotic cell is a cancer cell or has been targeted toa tumor.